Co-infection with hepatitis C virus (HCV) has emerged as a major cause of morbidity and mortality for persons living with HIV/AIDS. Epidemiologic studies clearly indicate that HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes. However, non-structural regions of the HCV genome such as NS5B are subject to distinct selection pressures - purifying selection necessary to preserve enzymatic functions critical for viral replication, drug resistance mutations, and/or compensatory mutations - compared to structural genome regions. Despite the growing importance of NS5B as an emerging target of HCV therapy, limited data are available regarding its genotypic variability and phenotypic properties in the absence of directly acting therapies. To address this substantial gap in our understanding of HCV replication and pathogenesis, we propose a comprehensive analysis of NS5B genotypic, phenotypic, and biochemical variability in two well-characterized populations with HCV and/or HIV co-infection. Such studies are critical to facilitate the rational design and utilization of novel HCV therapies and significantly improve the clinical management and overall survival of persons with chronic HCV infection.